Benzyloxyresorufin O-dealkylation Research Articles

Prokaryotic functional expression and activity comparison of three CYP9A genes from the polyphagous pest Helicoverpa armigera.

Cytochrome P450s (CYPs or P450s) have been long recognized as very important enzymes in the metabolism of xenobiotic and endogenous compounds, but only a few CYPs have been functionally characterized in insects. The effort in functional characterization of insect P450s is heavily hindered by technical difficulties in preparing active, individual P450 enzymes directly from the target insect. In this paper, we describe the functional expression of two additional pyrethroid resistance-associated CYP9A genes (CYP9A12 and CYP9A17) from the polyphagous pest Helicoverpa armigera in the facile Escherichia coli. The functionality of E. coli produced CYP9A12, CYP9A14, and CYP9A17 was investigated and activities of these CYP9As were compared against three probe substrates after reconstitution with NADPH-dependent cytochrome P450 reductase. The results showed that active forms of CYP9A12 and CYP9A17 were expressed in E. coli with a content of about 1.0-1.5 nmol mg-1 protein in membrane preparations. In vitro assays showed that CYP9A14 was capable of catalyzing O-dealkylation of methoxyresorufin (MROD), ethoxyresorufin (EROD), and benzyloxyresorufin (BROD), while CYP9A12 and CYP9A17 exhibited only MROD and EROD activities. Kinetic studies demonstrated that CYP9A14 had the greatest k cat/K m value for MROD, and CYP9A17 for EROD, while the lowest k cat/K m values for both MROD and EROD were observed for CYP9A12. The distinct biochemical traits suggest that the three paralogous CYP9As may play different roles in xenobiotic metabolism in this important pest.

P.3.c.005 - Potent inhibition of CYP1A2 and CYP2D6 by the novel antipsychotic drug asenapine in human liver

In this study, we performed a screening of the specificities of rat cytochrome P450 (CYP) isoforms for metabolic reactions known as the specific probes of human CYP isoforms, using 13 rat CYP isoforms expressed in baculovirus-infected insect cells or B-lymphoblastoid cells. Among the metabolic reactions studied, diclofenac 4-hydroxylation (DFH), dextromethorphan O-demethylation (DMOD) and midazolam 4-hydroxylation were specifically catalyzed by CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. These results suggest that diclofenac 4-hydroxylation, dextromethorphan O-demethylation and midazolam 4-hydroxylation are useful as catalytic markers of CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. On the other hand, phenacetin O-deethylation and 7-ethoxyresorufin O-deethylation were catalyzed both by CYP1A2 and by CYP2C6. Benzyloxyresorufin O-dealkylation and pentoxyresorufin O-dealkylation were also catalyzed by CYP1A2 in addition to CYP2B1. Bufuralol 1′-hydroxylation was extensively catalyzed by CYP2D2 but also by CYP2C6 and CYP2C11. p-Nitrophenol 2-hydroxylation and chlorzoxazone 6-hydroxylation were extensively catalyzed by CYP2E1 but also by CYP1A2 and CYP3A1. Therefore, it is necessary to conduct further study to clarify whether these activities in rat liver microsomes are useful as probes of rat CYP isoforms. In contrast, coumarin 7-hydroxylation and S- and R-mephenytoin 4′-hydroxylation did not show selectivity toward any isoforms of rat CYP studied. Therefore, activities of coumarin 7-hydroxylation and S- and R-mephenytoin 4′-hydroxylation are not able to be used as catalytic probes of CYP isoforms in rat liver microsomes. These results may provide useful information regarding catalytic probes of rat CYPs for studies using rat liver microsomal samples.

Peptide-Mediated Specific Immobilization of Catalytically Active Cytochrome P450 BM3 Variant.

Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is an interesting target for biotechnological applications, because of its vast substrate variety combined with high P450 monooxygenase activity. The low stability in vitro could be overcome by immobilization on surfaces. Here we describe a novel method for immobilization on metal surfaces by using selectively binding peptides. A P450 BM3 triple mutant (3M-P450BM3: A74G, F87V, L188Q) was purified as protein thioester and ligated to indium tin oxide or gold binding peptides (BP) named HighSP-BP and Cys-BP, respectively. The ligation products were characterized by Western Blot and tryptic digestion combined with mass spectrometry, and displayed high affinity binding on the depicted surfaces. Next, we could demonstrate by benzyloxyresorufin O-dealkylation assay (BROD assay) that the activity of immobilized ligation products is higher than for the soluble form. The study provides a new tool for selective modification and immobilization of P450 variants.

Application of a Continuous-Flow Bioassay to Investigate the Organic Solvent Tolerability of Cytochrome P450 BM3 Mutants

A novel methodology is presented to investigate the organic solvent tolerability of cytochrome P450 monooxygenase BM3 (CYP BM3) mutants. A fluorescence-based continuous-flow enzyme activity detection (EAD) setup was used to screen the activity of CYP BM3 mutants in the presence of organic solvents. The methodology is based on the CYP BM3–mediated O-dealkylation of benzyloxyresorufin to form the highly fluorescent product resorufin. The assay setup not only allows detection of the formed resorufin, but it also simultaneously monitors cofactor depletion online. The EAD setup was used to test the activity of a small library of novel CYP BM3 mutants in flow-injection analysis mode in the presence of the organic modifiers methanol, acetonitrile, and isopropanol. Mutants with enhanced tolerability toward all three solvents were identified, and the EAD setup was adapted to facilitate CYP BM3 activity screening against a gradient of an organic modifier to study the behavior of the small library of CYP BM3 mutants in more detail. The simple methodology used in this study was shown to be a very powerful tool to screen for novel CYP BM3 mutants with increased tolerability toward organic solvents.

Enzymatic characterization of in vitro-expressed Baikal seal cytochrome P450 (CYP) 1A1, 1A2, and 1B1: Implication of low metabolic potential of CYP1A2 uniquely evolved in aquatic mammals

This study aimed to elucidate the catalytic function of cytochrome P450 (CYP) 1 enzymes in aquatic mammals. Alkoxyresorufin O-dealkylation (AROD) activities including methoxy- (MROD), ethoxy- (EROD), pentoxy- (PROD), and benzyloxyresorufin O-dealkylation (BROD), and 2- and 4-hydroxylation activities of 17β-estradiol (E2) were measured by using yeast-expressed Baikal seal (Pusa sibirica) CYP1A1, 1A2, and 1B1 proteins. Heterologous protein expression of the Baikal seal CYP1s (bsCYP1s) in yeast microsomes was confirmed by reduced CO-difference spectra and immunoblotting. Heterologously expressed human CYP1 enzyme (hCYP1) activities were simultaneously measured and compared with those of bsCYP1 isozymes. Recombinant bsCYP1A1 protein showed the highest Vmax of EROD, followed by MROD, PROD, and BROD, similar to that of hCYP1A1. Vmax/Km ratios of all AROD activities catalyzed by bsCYP1A1 were lower than those catalyzed by hCYP1A1, suggesting less potential for AROD by bsCYP1A1. Enzymatic assays for bsCYP1A2 showed no or minimal AROD activities, while hCYP1A2 displayed MROD and EROD activities. bsCYP1B1 showed an AROD profile (EROD>BROD>MROD>>PROD) similar to that of hCYP1B1; however, Vmax/Km ratios of all AROD activities by bsCYP1B1 were higher. Yeast microsomes containing bsCYP1A1 and 1B1 and hCYP1A1, 1A2, and 1B1 metabolized E2 to 2-OHE2 and 4-OHE2, whereas bsCYP1A2 showed no such activity. Comparison of 4- and 2-hydroxylations of E2 by CYP1As suggests that bsCYP1A1, hCYP1A1, and 1A2 preferentially catalyze 2- rather than 4-hydroxylation. As for CYP1B1, the Vmax/Km ratios suggest that both Baikal seal and human CYPs catalyze 4- rather than 2-hydroxylation. Interspecies comparison showed that bsCYP1B1 has higher metabolic potencies for both E2 hydroxylations than does hCYP1B1, whereas the activity of bsCYP1A1 was lower than that of hCYP1A1. Messenger RNA expression levels of bsCYP1s in the liver of Baikal seals indicated that bsCYP1A1 and 1A2 enzymes contributed to 16.2% and 83.7% of total CYP1s, respectively; bsCYP1B1 accounted for only 0.06%. Addition of anti-human CYP1A1 antibody in seal liver microsomes suppressed EROD activity more than did anti-human CYP1A2 antibody. Therefore, EROD may be catalyzed by hepatic bsCYP1A1 but not bsCYP1A2, consistent with the results of yeast-expressed bsCYP1A1 and 1A2. In silico substrate-docking models of bsCYP1s suggested that the defect in bsCYP1A2 enzymatic activities may be accounted for by the Pro substitution of highly conserved Thr in the I-helix, which is involved in formation of a hydrogen bond with the hydroperoxy intermediate on the heme. This Thr-Pro substitution is evolutionarily conserved across aquatic mammals and could explain their lower metabolic potential for persistent organic pollutants.

Different profiles of hepatic alkoxyresorufin O‐dealkylase activities in small rodents

The aims of the present study were to determine cytochrome P450 enzyme activity in six strains of experimental rodents (n = 5/sex/species): ICR, C57BL/6 and DBA/2 mice; Sprague Dawley and Wistar rats; and Dunkin Hartley guinea pigs. After animals were treated with the typical inducers β-naphthoflavone (BNF), dexamethasone (DEX) and phenobarbital (PB), the levels of O-dealkylation of ethoxyresorufin (EROD), methoxyresorufin (MROD), pentoxyresorufin (PROD) and benzyloxyresorufin (BROD) activity were determined using responsive catalytic reactions to study CYP1A1, CYP1A2 and CYP2B, respectively. A maximal induction of EROD and MROD was found in BNF-treated animals from all strains (2.4- to 15.1-fold) except DBA/2 (0.9- to 1.8-fold). C57BL/6 mice had the strongest BNF-induced EROD (15.1-fold) and MROD (8.3-fold) activities. No differences in BNF-induced EROD and MROD activities were observed between males and females. However, the EROD activity of Wistar rats and the MROD activity of Sprague Dawley rats were higher in males than females. DEX induced PROD activity only in mice (1.3- to 7.1-fold), but not in rats and guinea pigs (0.2- to 1.1-fold). However, induction of BROD activity was found in DEX-treated mice and rats (1.5 to 12.5-fold), but not in guinea pigs (0.3 to 0.4-fold). PB caused a significant elevation of PROD (1.7- to 10.4-fold) and BROD (31- to 13.2-fold) activities in all the animals. PB-induced BROD activity was higher in females than males in Sprague Dawley rats. These observations strongly suggest that the choice of experimental animal strain, species and inducer is of critical importance for studies of drug metabolism and interaction.

Species-Dependent and Receptor-Selective Action of Bilobalide on the Function of Constitutive Androstane Receptor and Pregnane X Receptor

Bilobalide is a naturally occurring sesquiterpene trilactone with therapeutic potential in the management of ischemia and neurodegenerative diseases such as Alzheimer's disease. In the present study, we investigated the effect of bilobalide on the activity of rat constitutive androstane receptor (rCAR) and rat pregnane X receptor (rPXR) and compared that with human CAR (hCAR) and human PXR (hPXR). Bilobalide activated rCAR in a luciferase reporter gene assay and increased rCAR target gene expression in cultured rat hepatocytes, as determined by the CYP2B1 mRNA and CYP2B enzyme activity (benzyloxyresorufin O-dealkylation) assays. This increase in hepatocyte CYP2B1 expression by bilobalide was not accompanied by a corresponding increase in rCAR mRNA level. In contrast to the activation of rCAR, the activity of rPXR, hCAR, and hPXR was not influenced by this chemical in cell-based reporter gene assays. Consistent with these results, bilobalide did not alter rPXR, hCAR, or hPXR target gene expression in rat or human hepatocytes, as evaluated by the CYP3A23, CYP2B6, CYP3A4 mRNA assays and the CYP3A (testosterone 6β-hydroxylation) and CYP2B6 (bupropion hydroxylation) enzyme activity assays. Bilobalide was not an antagonist of rPXR, hCAR, or hPXR, as suggested by the finding that it did not attenuate rPXR activation by pregnenolone 16α-carbonitrile, hCAR activation by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime, or hPXR activation by rifampicin in reporter gene assays. In conclusion, bilobalide is an activator of rCAR, whereas it is not a ligand of rPXR, hCAR, or hPXR. Likewise, it is an inducer of rat CYP2B1, but not of rat CYP3A23, human CYP2B6, or human CYP3A4.

Biotransformation of PCBs in Relation to Phase I and II Xenobiotic-Metabolizing Enzyme Activities in Ringed Seals (Phoca hispida) from Svalbard and the Baltic Sea

Polychlorinated biphenyls (PCBs) may induce activity of hepatic enzymes, mainly Phase I monooxygenases and conjugating Phase II enzymes, that catalyze the metabolism of PCBs leading to formation of metabolites and to potential adverse health effects. The present study investigates the concentration and pattern of PCBs, the induction of hepatic phase I and II enzymes, and the formation of hydroxy (OH) and methylsulfonyl (CH3SO2=MeSO2) PCB metabolites in two ringed seal (Phoca hispida) populations, which are contrasted by the degree of contamination exposure, that is, highly contaminated Baltic Sea (n=31) and less contaminated Svalbard (n=21). Phase I enzymes were measured as ethoxyresorufin-O-deethylation (EROD), benzyloxyresorufin-O-dealkylation (BROD), methoxyresorufin-O-demethylation (MROD), and pentoxyresorufin-O-dealkylation (PROD) activities, and phase II enzymes were measured as uridine diphosphophate glucuronosyl transferase (UDPGT) and glutathione-S-transferase (GST). Geographical comparison, multivariate, and correlation analysis indicated that sigma-PCB had a positive impact on Phase I enzyme and GST activities leading to biotransformation of group III (vicinal ortho-meta-H atoms and < or =1 ortho-chlorine (Cl)) and IV PCBs (vicinal meta-para-H atoms and < or =2 ortho-Cl). The potential precursors for the main OH-PCBs detected in plasma in the Baltic seals were group III PCBs. MeSO2-PCBs detected in liver were mainly products of group IV PCB metabolism. Both CYP1A- and CYP2B-like enzymes are suggested to be involved in the PCB biotransformation in ringed seals.

Purification of CYP2B‐like protein from feral leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular, and immunological characterization

In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M(r) of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time.

Catalytic function of avian cytochrome P450 1A4 and 1A5 (CYP1A4 and CYP1A5) enzymes heterologously expressed using in vitro yeast system

The present study clarifies the enzymatic properties of two avian cytochrome P4501A (CYP1A) paralogs, CYP1A4 and 1A5, using a yeast-based vector system. Recombinant CYP1A4 and 1A5 proteins from common cormorant ( Phalacrocorax carbo) were expressed in yeast cells, and showed typical reduced CO-difference spectra with a peak at 446 nm. Kinetic analysis of O-dealkylase of methoxy-, ethoxy-, pentoxy- and benzyloxyresorufin catalyzed by the CYP1A enzymes revealed that V max value for ethoxyresorufin- O-deethylase (EROD) activity was much higher than that for the other three O-dealkylase activities for both isozymes. Interestingly, remarkable substrate specificity of the CYP1As was observed for O-dealkylation of benzyloxyresorufin and methoxyresorufin; CYP1A4 was highly specific for catalyzing benzyloxyresorufin- O-debenzylase activity, whereas CYP1A5 was more efficient in catalyzing methoxyresorufin- O-demethylase activity. The present study also measured CYP1A-dependent EROD activity in the presence of 2,3,7,8-tetrachlorodibenzofuran (TCDF) to evaluate the ability of this dioxin-like congener to inhibit the EROD activity. One hundred nanomolar TCDF noncompetitively inhibited CYP1A5-dependent EROD activity, although no inhibitory effect was detected for CYP1A4-dependent EROD activity. These results indicate that the avian CYP1A paralogs have different affinities for substrate and inhibitor, thus suggesting their distinct physiological and toxicological roles.

Fluconazole-induced hepatic cytochrome P450 gene expression and enzymatic activities in rats and mice

This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague–Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods were used as measures of Cyp enzyme activities. Western analyses identified specific Cyp isoforms. Quantitative real-time reverse-transcription polymerase chain reaction (quantitative real time-RT-PCR) assays were used to quantitate the mRNA expression of specific Cyp genes induced by this conazole. Rats and mice were administered fluconazole 2, 25, or 50 mg/kg bw/d by gavage daily for 14 days. In rats, fluconazole treatment (50 mg/kg bw/d) significantly induced pentoxyresorufin O-dealkylation (PROD), benzyloxyresorufin O-dealkylation (BROD), and ethoxyresorufin O-dealkylation (EROD) hepatic microsomal activities. Fluconazole treatment significantly increased rat hepatic mRNA expression of CYP2B1 and CYP3A23/3A1 with dose-related responses. The highest dose of fluconazole gave a 128-fold induction of CYP2B1 and a 4.6-fold induction of CYP3A23/3A1 mRNA. CYP3A2 mRNA levels were also overexpressed 5.6–7.2-fold depending on dose. Western immunoblots of rat hepatic microsomal proteins identified Cyp isoforms: CYP1A1, CYP1A2, CYP2B1/2, CYP3A23/3A1, and Cyp3A2 with increased levels of CYP2B1/2 and CYP3A23/3A1 proteins. In mice, fluconazole induced BROD, PROD, EROD, and methoxyresorufin O-dealkylation hepatic microsomal activities after treatment with 25 and 50 mg/kg bw/d. Fluconazole increased mouse hepatic mRNA expression of Cyp2b10 (1.9-fold) and Cyp3a11 (2.6-fold) in the 50 mg/kg bw/d treatment group. In summary, these results indicated that fluconazole, a triazole-containing conazole, clearly induced CYP2B and CYP3A families of isoforms in rat liver and Cyp2b and Cyp3a families of isoforms in mouse liver.

Evaluation of alkoxyresorufins as fluorescent substrates for cytochrome P450 BM3 and site-directed mutants

In this study, the first fluorescent assay for bacterial cytochrome P450 BM3 (BM3) and mutants is described. BM3 mutants are potentially very versatile biocatalysts for the production of fine chemicals. A fluorescent assay would be very useful for the identification of nonnatural ligands in high-throughput inhibition assays. Because of the ease and sensitivity of alkoxyresorufin O-dealkylation assays, four different alkoxyresorufins were evaluated as substrates. Wild-type BM3 showed extremely low activity toward all four alkoxyresorufins tested. Five different BM3 mutants were constructed, carrying different combinations of mutations R47L, F87V, and L188Q, which were previously shown to increase activity toward nonnatural substrates. For all mutants, a high benzyloxyresorufin O-dealkylation (BROD) activity was found. The triple mutant of BM3, R47L/F87V/L188Q, showed the highest activity, increasing 900-fold compared to wild-type BM3. The BROD assay could also be applied in whole Escherichia coli cells; permeabilization by lipopolysaccharide deficiency strongly increased activity. To demonstrate the applicability of the BROD assay to screening for novel ligands of BM3 R47L/F87V/L188Q, a library of 45 drug-like compounds was tested for inhibition. Of these compounds, 8 showed strong inhibition of the BROD activity, demonstrating for the first time that drug-like molecules also can bind with high affinity to BM3 mutants.

Substrate specificity for rat cytochrome P450 (CYP) isoforms: screening with cDNA-expressed systems of the rat

In this study, we performed a screening of the specificities of rat cytochrome P450 (CYP) isoforms for metabolic reactions known as the specific probes of human CYP isoforms, using 13 rat CYP isoforms expressed in baculovirus-infected insect cells or B-lymphoblastoid cells. Among the metabolic reactions studied, diclofenac 4-hydroxylation (DFH), dextromethorphan O-demethylation (DMOD) and midazolam 4-hydroxylation were specifically catalyzed by CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. These results suggest that diclofenac 4-hydroxylation, dextromethorphan O-demethylation and midazolam 4-hydroxylation are useful as catalytic markers of CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. On the other hand, phenacetin O-deethylation and 7-ethoxyresorufin O-deethylation were catalyzed both by CYP1A2 and by CYP2C6. Benzyloxyresorufin O-dealkylation and pentoxyresorufin O-dealkylation were also catalyzed by CYP1A2 in addition to CYP2B1. Bufuralol 1′-hydroxylation was extensively catalyzed by CYP2D2 but also by CYP2C6 and CYP2C11. p-Nitrophenol 2-hydroxylation and chlorzoxazone 6-hydroxylation were extensively catalyzed by CYP2E1 but also by CYP1A2 and CYP3A1. Therefore, it is necessary to conduct further study to clarify whether these activities in rat liver microsomes are useful as probes of rat CYP isoforms. In contrast, coumarin 7-hydroxylation and S- and R-mephenytoin 4′-hydroxylation did not show selectivity toward any isoforms of rat CYP studied. Therefore, activities of coumarin 7-hydroxylation and S- and R-mephenytoin 4′-hydroxylation are not able to be used as catalytic probes of CYP isoforms in rat liver microsomes. These results may provide useful information regarding catalytic probes of rat CYPs for studies using rat liver microsomal samples.

Enhancement of N-Nitrosodiethylamine-lnitiated Hepatocarcinogenesis by Phenytoin in Male F344/NCr Rats at a Dose Causing Maximal Induction of CYP2B

The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of male F344/NCr rats initiated with a single i.p. dose of N-nitrosodiethylamine (75 mg/kg b.w.) was studied. Beginning 2 weeks post-initiation, the rats received control diet or diet containing 500 or 1500 ppm DPH or 500 ppm phenobarbital. At 52 weeks age, the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0%, 17% (1 ± 0), 42% (1.8 ± 0.8), or 67% (2.5 ± 1.9) in rats exposed to N-nitrosodiethylamine alone, or the carcinogen followed by 500 ppm DPH, 1500 ppm DPH, or 500 ppm phenobarbital, respectively. Between 53 and 79 weeks of age, 39% of rats receiving N-nitrosodiethylamine alone developed multiple (1.5 ± 0.8) hepatocellular adenomas. A similar incidence (41%) occurred in the rats administered the carcinogen followed by 500 ppm DPH. The incidence of hepatocellular adenomas (88% and 89%) was significantly greater in rats exposed to N-nitrosodiethylamine followed by 1500 ppm DPH or 500 ppm phenobarbital, respectively. Multiplicities of hepatocellular adenomas were significantly greater than the control value in rats fed 1500 ppm DPH or 500 ppm phenobarbital (5.9 ± 4.8 and 10.1 ± 6.7, respectively), but not in the rats receiving 500 ppm DPH (2.3 ± 1.6). No rats exposed to N-nitrosodiethylamine alone or the carcinogen followed by 500 ppm DPH developed hepatocellular carcinomas, while hepatocellular carcinomas occurred in 29% or 67% of the rats given 1500 ppm DPH or 500 ppm phenobarbital, respectively, following initiation. Increases in hepatic CYP2B-mediated benzyloxyresorufin O-dealkylation activity in rats exposed to 500 and 1500 ppm DPH for 2 or 23 weeks were ∼50% and ∼100%, respectively, of the maximal induction caused by 500 ppm phenobarbital. Thus, in the rat model, DPH enhanced N-nitrosodiethyl-amine-initiated hepatocarcinogenesis when administered at a dose causing maximal CYP2B induction.

Enhancement of N-nitrosodiethylamine-initiated hepatocarcinogenesis by phentoin in male F344/NCr rats at a dose causing maximal induction of CYP2B.

The effect of the clinically important anticonvulsant phenytoin (DPH) on hepatocarcinogenesis of male F344/NCr rats initiated with a single i.p. dose of N-nitrosodiethylamine (75 mg/kg b.w.) was studied. Beginning 2 weeks post-initiation, the rats received control diet or diet containing 500 or 1,500 ppm DPH or 500 ppm phenobarbital. At 52 weeks age, the incidences (and multiplicities, in units of tumors per tumor-bearing rat) of hepatocellular adenomas were 0%, 17% (1 +/- 0), 42% (1.8 +/- 0.8), or 67% (2.5 +/- 1.9) in rats exposed to N-nitrosodiethylamine alone, or the carcinogen followed by 500 ppm DPH, 1,500 ppm DPH, or 500 ppm phenobarbital, respectively. Between 53 and 79 weeks of age, 39% of rats receiving N-nitrosodiethylamine alone developed multiple (1.5 +/- 0.8) hepatocellular adenomas. A similar incidence (41%) occurred in the rats administered the carcinogen followed by 500 ppm DPH. The incidence of hepatocellular adenomas (88% and 89%) was significantly greater in rats exposed to N-nitrosodiethylamine followed by 1,500 ppm DPH or 500 ppm phenobarbital, respectively. Multiplicities of hepatocellular adenomas were significantly greater than the control value in rats fed 1,500 ppm DPH or 500 ppm phenobarbital (5.9 +/- 4.8 and 10.1 +/- 6.7, respectively), but not in the rats receiving 500 ppm DPH (2.3 +/- 1.6). No rats exposed to N-nitrosodiethylamine alone or the carcinogen followed by 500 ppm DPH developed hepatocellular carcinomas, while hepatocellular carcinomas occurred in 29% or 67% of the rats given 1,500 ppm DPH or 500 ppm phenobarbital, respectively, following initiation. Increases in hepatic CYP2B-mediated benzyloxyresorufin O-dealkylation activity in rats exposed to 500 and 1,500 ppm DPH for 2 or 23 weeks were approximately 50% and approximately 100%, respectively, of the maximal induction caused by 500 ppm phenobarbital. Thus, in the rat model, DPH enhanced N-nitrosodiethyl-amine-initiated hepatocarcinogenesis when administered at a dose causing maximal CYP2B induction.

CYP3A4 drug interactions: correlation of 10 in vitro probe substrates.

Many substrates of cytochrome P450 (CYP) 3A4 are used for in vitro investigations of drug metabolism and potential drug-drug interactions. The aim of the present study was to determine the relationship between 10 commonly used CYP3A4 probes using modifiers with a range of inhibitory potency. The effects of 34 compounds on CYP3A4-mediated metabolism were investigated in a recombinant CYP3A4 expression system. Inhibition of erythromycin, dextromethorphan and diazepam N-demethylation, testosterone 6beta-hydroxylation, midazolam 1-hydroxylation, triazolam 4-hydroxylation, nifedipine oxidation, cyclosporin oxidation, terfenadine C-hydroxylation and N-dealkylation and benzyloxyresorufin O-dealkylation was evaluated at the apparent Km or S50 (for substrates showing sigmoidicity) value for each substrate and at an inhibitor concentration of 30 microM. While all CYP3A4 probe substrates demonstrate some degree of similarity, examination of the coefficients of determination, together with difference and cluster analysis highlighted that seven substrates can be categorized into two distinct substrate groups. Erythromycin, cyclosporin and testosterone form the most closely related group and dextromethorphan, diazepam, midazolam and triazolam form a second group. Terfenadine can be equally well placed in either group, while nifedipine shows a distinctly different relationship. Benzyloxyresorufin shows the weakest correlation with all the other CYP3A4 probes. Modifiers that caused negligible inhibition or potent inhibition are generally comparable in all assays, however, the greatest variability is apparent with compounds causing, on average, intermediate inhibition. Modifiers of this type may cause substantial inhibition, no effect or even activation depending on the substrate employed. It is recommended that multiple CYP3A4 probes, representing each substrate group, are used for the in vitro assessment of CYP3A4-mediated drug interactions.

Induction of Hepatic Cytochromes P450 by Dietary 1,4-Bis[2–(3,5–dichloropyridyloxy)] benzene (TCPOBOP) in the B6C3F1 Mouse: Dose-Dependence and Pharmacodynamics

The dose-response relationships for hepatic cytochrome P450 (CYP) induction by 1,4-bis(2-[3,5-dichloropyridyloxy])benzene (TCPOBOP) were examined in the male B6C3F1 mouse. TCPOBOP, administered for 14 days at 0.393–96.2 ppm in the diet, caused concentration-dependent induction of hepatic CYP subfamily 2B (C YP2B) protein and of benzyloxy resorufin O-dealkylation. The ED50 values for CYP2B induction, calculated on the basis of immunoreactiveCYP2B protein and benzyloxyresorufln O-dealkylation, were 1.2 ppm and 2.2 ppm dietary TCPOBOP, respectively. The maximal catalytic activities observed were 74–95–fold higher than control values. These induction levels were 2.3–2.6–fold greater than those resulting from exposure of the mice to 500 ppm dietary phenobarbital (a prototypic CYP2B inducer). The EC50 values of TCPOBOP for hepatic CYP2B induction were &lt; 0.3 μmol/1 (serum) and &lt;3.3 μmol/kg (liver). Increases in immunoreactive CYP subfamily 1A and subfamily 3A proteins were also detected, although the maximal induction levels for these subfamilies were relatively low (&lt; threefold, compared to control values). These results indicate that TCPOBOP is an effective CYP2B inducer in the mouse when administered at dietary concentrations well below the no-effect level (12.3 ppm) for the rat.

Isoform selective inhibition and inactivation of human cytochrome P450s by methylenedioxyphenyl compounds

1. A series of methylenedioxyphenyl compounds were evaluated for their inhibitory and inactivation effects on nine human cytochrome P450 (CYP) activities using microsomes from human B-lymphoblast cells expressing specific human CYP isoforms. 2. Methylenedioxyphenyl compounds which possess a bulky structure such as 1,4- benzothiazine showed substantial inhibition of S-warfarin 7-hydroxylation catalysed by CYP2C9, S-mephenytoin 4'-hydroxylation by CYP2C19, bufuralol 1-hydroxylation by CYP2D6, and testosterone 6β-hydroxylation by CYP3A4. Regarding ethoxyresorufin O-deethylation catalysed by CYP1A1 and benzyloxyresorufin O-dealkylation by CYP2B6, the subtle change of a substitution of the 1,4-benzothiazine structure affected the inhibition selectivity. Ethoxyresorufin O-deethylation by CYP1A2, coumarin 7-hydroxylation by CYP2A6, and chlorzoxazone 6-hydroxylation by CYP2E1 were not inhibited by almost any of the methylenedioxyphenyl compounds. The inhibitory effects of methylenedioxyphenyl compounds that possess a short chain amino group on the human CYP isoforms were not significant. 3. The methylenedioxyphenyl compounds inactivated CYP1A1 (kinact=0.034 min-1 and Ki=0.81 μM), CYP2C9 (kinact=0.041 and 0.042 min-1 and Ki=0.56 and 0.15 μM), CYP2D6 (kinact=0.044-0.339 min-1 and Ki=0.21-19.88 μM), and CYP3A4 (kinact=0.076-0.251 min-1 and Ki=0.25-0.69 μM). These results suggested that the methylenedioxyphenyl compounds investigated in this study would be potent mechanismbased inactivators of these human CYP isoforms. In contrast, CYP2B6 and CYP2C19 were not inactivated. 4. The present study suggested that the selectivity of inhibition or inactivation of human CYP isoforms by methylenedioxyphenyl compounds may vary according to the structure of the side chain.

Dose-response relationships for cytochrome P450 induction by phenobarbital in the cotton rat ( Sigmodon hispidus)

The induction of a hepatic pleiotropic response, including increase in liver/body weight ratio, induction of hepatic CYP2B and CYP3A protein and catalytic activity, and hepatic microsomal epoxide hydration activity, was investigated in male cotton rats ( Sigmodon hispidus) administered graded dietary concentrations (0–1500 ppm) of phenobarbital (PB) for 14 days. A dose-dependent induction of each endpoint was observed, although plateaus in the various dose-response curves were not obtained, and ED 50 values (PB concentrations associated with half-maximal responses) for the various endpoints were not able to be calculated. A maximal 1.31-fold increase, compared to the control value, in liver/body weight ratio was observed, while microsomal epoxide hydration activity was increased as much as 3.6-fold by PB administration. Pentoxy- and benzyloxyresorufin O-dealkylation and testosterone 16 β-hydroxylation activities (considered to be relatively selective for CYP2B in the Norway rat ( Rattus norvegicus)), were induced maximally less than five-fold. Testosterone 6 β-hydroxylation (considered to be relatively selective for CYP3A in R. norvegicus) was induced maximally less than two-fold. Maximal induction of 7-ethoxy-4-trifluoromethyl-coumarin O-deethylation was 18-fold, compared to the control rate. Western blotting studies indicated that hepatic microsomal proteins immunoreactive with polyclonal antisera to R. norvegicus CYP2B1 or CYP3A1 were induced, in a dose-responsive manner, by PB in the cotton rats. These results indicate that the cotton rat responds to PB treatment with a coordinate pleiotropic response similar to that displayed by R. norvegicus, although the substrate specificity of the induced proteins appears to differ between the two rodent species.

Induction of liver monooxygenases by β-myrcene

β-Myrcene (MYR) is an acyclic monoterpene found in the essential oils of a variety of useful plants such as lemongrass ( Cymbopogon citratus), hop, verbena, bay and others. MYR and essential oils containing this olefinic monoterpene are widely used as flavoring food additives, as fragrances in cosmetics and as scents in household products. The present study was undertaken to investigate the induction of liver monooxygenases by MYR. Female Wistar rats were treated by gavage with MYR (1000 mg/kg body weight) or corn oil (vehicle) for 1 or 3 consecutive days. Activities of ethoxycoumarin- O-deethylase (ECOD) and alkoxy-resorufin O-dealkylases (methoxy- (MROD), ethoxy- (EROD), pentoxy- (PROD) and benzyloxy-resorufin- O-dealkylation (BROD)) were determined fluorimetrically in the hepatic microsomal fraction. Exposure to MYR, either for 1 or 3 days, produced marked (13–34-fold) increases in the activities of PROD and BROD and only minor changes in ECOD, EROD and MROD. Since PROD and BROD are metabolized mainly by CYP2B isoenzymes, these results suggest that MYR induces this phenobarbital-inducible P450 subfamily. The induction of CYP2B isoenzymes was confirmed by SDS-PAGE and immunoblotting. Levels of apoproteins CYP2B1/2B2 were increased 8.2-fold after treatment with MYR (1000 mg/kg body wt, 3 days). Results from this study therefore indicate that MYR is an inducer of isoenzymes belonging to CYP2B subfamily.